Isolation and Identification of E. cowanii from Powdered Infant Formula in NICU and Determination of Antimicrobial Susceptibility of Isolates.

OBJECTIVE
Enterobacter cowanii is a genus of common gram-negative, facultatively anaerobic, rod-shaped, non-spore-forming bacterium of the Enterobacteriaceae family. This organism can be potentially a powdered infant milk formula-borne opportunistic pathogen. The aim of this study was to isolate and identify E. cowanii from consumed powdered infant formula milk (PIF) in intensive care units (NICU) and to determine antimicrobial susceptibility patterns of this bacterium.


METHODS
E. cowanii was isolated according to FDA method in 125 samples of PIF milk purchased from drug stores between Jun 2011 and March 2012. For final confirmation, biochemical tests embedded in API-20E system were used. The drug susceptibility test was performed using the disc diffusion method according to CLSI recommendations. Findings : Out of the 125 PIF samples investigated, 4 (3.2%) samples were positive for E. cowanii. All four isolates from PIF samples were uniformly susceptible to imipenem, meropenem, ceftazidime, ciprofloxacin, and colistin. Fifty percent of isolates were resistant to ampicillin, amoxicillin, and cotrimoxazole Conclusion: Analysis of the results indicated that complementary studies are necessary to clarify the possible role of E. cowanii as a food contaminant, in common NICU infections and high risk groups including persons with underlying disease and immunocompromised individuals.


Introduction
The genus Enterobacter in a family of Enterobacteriaceae was first described by Hormaeche and Edwards in 1960 and has undergone considerable taxonomic modification over the last 50 years [1,2] . The name Enterobacter cowanii is proposed for a group of microorganisms referred to as NIH group 42. The G + C content of its DNA ranges from 52.5% to 53.6% [3,4] . Because of distinct differentiation of E. cowanii by DNA hybridization methods from other members of Enterobacteriaceae family as well as its unique phenotypic and genotypic properties and since the DNA relatedness (5-38%) is closer to species of the genus Enterobacter than to other species of the Enterobacteriaceae, the members of NIH group 42 were placed in the genus Enterobacter [3][4][5] . The majority of E. cowanii strains were isolated from clinical and plant specimens [3] .
E. cowanii is a genus of common gram-negative, facultatively anaerobic, rod-shaped, oxidasenegative, catalase positive, non-spore-forming bacterium of the Enterobacteriaceae family. It is

Original Article Iran J Pediatr Jun 2014; Vol 24 (No 3), Pp: 261-266
motile by peritrichous flagella [2,4] . This organism grows well on selective media for gram negative bacteria such as MacConkey agar and is facultatively anaerobic. E. cowanii is commonly found in ecological niches. This opportunistic pathogen is isolated from clinical specimens such as urine, sputum, blood, and pus. It was isolated by blood culture from a patient with gastric cancer. It may also be found in foods [4,5] . E. cowanii has been isolated from eucalyptus trees [6,7] .
In recent years a remarkable increase in nosocomial infections has been reported especially in neonatal intensive care units, intensive care units and oncology departments. Underlying diseases, low-birth-weight, immunocompromised immune system, cancer chemotherapy, and intravenous catheterization can be predisposing factors in cases of infections due to unusual microorganisms, including Enterobacter spp. in neonates [8][9][10][11] . Infants admitted to NICUs, especially ones who have undergone surgery or have congenital abnormalities, are often at high risk for developing nosocomial infections. Clinical manifestations are often misleading and, in some circumstances, it may be difficult or even impossible to distinguish the source of the infection [9,10] . Enterobacteriaceae family members are potential PIF-borne pathogens.
Neonates and young children are exclusively vulnerable to infections caused by foodborne pathogens [9,12] . Contamination of PIF with E. cowanii will be associated with many diseases in neonates. Therefore, the microbiological safety of PIF is very important. Because PIF is not a sterile product, it is an excellent medium to support the bacterial growth. Bovine milk and plant materials are essential ingredients of PIF and a potential source of various bacteria that are pathogenic to neonates and adults [12][13][14] . The aim of our study was to isolate and identify antimicrobial susceptibility pattern of E. cowanii isolated from PIF in NICUs in Tehran hospitals.

Subjects and Methods
Place and Duration of Study: Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, between Jun 2011 and March 2012. Sampling: A cross-sectional study was carried out on 125 samples of powdered infant formula milk (PIF) purchased from hospital drug stores in Tehran between Jun 2011 and March 2012. Isolation and Identification: PIF cans were surface sanitized with 70% ethanol and were opened in a laminar flow cabinet. Samples were taken from each product under aseptical conditions. E. cowanii was isolated according to FDA method [15,16] . We prepared 3 Erlenmeyer flasks of sterile distilled water (pre-warmed to 45ºC) at 9, 90 and 900 ml containing 1, 10 and 100 g of PIF, respectively. After the PIF was completely mixed and dissolved in distilled water, it was incubated at 37ºC for 18-24 h. Following incubation, 10 ml of each sample was added to 90 ml of Enterobacteriaceae enrichment (EE) broth medium and incubated at 37ºC for 18-24 h. After incubation, a lapful of the enrichment culture was streaked onto duplicates violet red bile glucose agar (VRBGA) plates and cultured at 37ºC for 18-24 h. A total of 4 suspicious colonies were picked from each VRBGA plate and pure culture was achieved. For detection of non-lactose fermenting isolates, presumptive colonies were streaked onto MacConkey agar and incubated at 37ºC for 72 h. For final confirmation biochemical tests were embedded in the API-20E biochemical kit system (Bio-Mérieux) and manual biochemical tests were used according to directions of the manufacturer. For long term storage, the purified isolates were stored in tryptic soy broth (TSB) with 20% glycerol (Merck Co.) at -20ºC. Antibiotic sensitivity testing: Antibiotic sensitivity testing was performed using Kirby-Bauer disk diffusion method on Mueller Hinton agar according to CLSI guidelines [17] . Antimicrobial agents used in this study are listed in Table 1. Statistical analysis: The calculation of sample size was performed by using McNemar's test. Data were analyzed using SPSS software, version 19.
The gram staining of the colony of organism showed gram negative rods. On VRBGA agar selective medium purple/pink colored colonies, and on MacConkey agar lactose fermenting, smooth, convex, punctuate, umbilicated, glistening colonies were grown during 16 to 20 hours. Isolated strains were oxidase negative, catalase positive, motile and produced other biochemical reactions which are characteristic of E. cowanii (Table 2). Fifty percent of isolates were resistant to ampicillin, amoxicillin, and cotrimoxazole. Susceptibility patterns of isolates are listed in Table 1.

Discussion
E. cowanii shows the common characteristics of the genus Enterobacter, mostly isolated from different sources such as clinical specimens, foods, plants, in developed and developing countries worldwide [3][4][5][6] . One large difficulty in preventing NICU and hospital-acquired infections is to find the source of the infectious agent and its route of transmission. The consumption of powdered infant milk formula is wide-spreading; however, few studies have been undertaken to evaluate the role of E. cowanii in food safety.
To the best of our knowledge, there is no previous report on the isolation and identification of E. cowanii from PIF and determination of antimicrobial susceptibility pattern of this bacterium in Iran. In this study, we demonstrated that E. cowanii strains are widely spread in PIF. The results showed that all E. cowanii strains isolated from PIF samples were sensitive to meropenem, ceftazidime, ciprofloxacin, imipenem, chloram-phenicol, cefepime, levofloxacin, piperacillin, gentamicin, moxifloxacin, and colistin. In the present study 50% of isolates were resistant to ampicillin, amoxicillin, and cotrimoxazole.
To the best of our knowledge, there is no previous report on the isolation and identification of E. cowanii from PIF and determination of antimicrobial susceptibility pattern of this bacterium in Iran. In this study, we demonstrated that E. cowanii strains are widely spread in PIF. The results showed that all E. cowanii strains isolated from PIF samples were sensitive to meropenem, ceftazidime, ciprofloxacin, imipenem, chloram-phenicol, cefepime, levofloxacin, piperacillin, gentamicin, moxifloxacin, and colistin. In the present study 50% of isolates were resistant to ampicillin, amoxicillin, and cotrimoxazole.
E. cowanii is an opportunistic organism and, when introduced into the human organs or other fauna, may cause infection. Disease caused by this organism can occur in individuals with underlying diseases especially patients hospitalized in NICU [10] . Confirmed virulence of E. cowanii is difficult to reveal, because clinical reports involving E. cowanii are typically of polymicrobial nature, often involve patients that are already affected by diseases of other origin, lack pathogenicity confirmation, and diagnostic isolates are rarely conserved for confirmatory analysis. The results of the present study helps to better understanding of the role of E. cowanii as an opportunistic pathogen-causing disease in NICU.
Neonates and high risk groups (e.g. immunosuppressed and HIV positive individuals, senile persons) are particularly assailable to foodborne opportunistic pathogens [18,19] . Low birth weight infants in neonatal intensive care units are typically immunocompromised patients and their immunity is not fully mature. Therefore they are susceptible to different hospital-acquired infections [10,[20][21][22] . In newborn infants, local natural barriers against bacterial infections are compromised and the production of secretory immunoglobin A is absent during the first days of life [9] . The decreased production and function of local and systemic defense depends on antigen exposure and contributes to greater susceptibility to bacterial infection during the neonatal period [9,23] . From our results we conclude that E. cowanii may be able to start a hospital outbreak, through powdered infant milk formula. The inherent capability of this organism to remain viable and grow well at room temperature may contribute to such contamination. Caregivers in hospital neonatal units should be constantly alerted to the fact that powdered infant formula products are not sterile and may be colonized with different microorganisms.
In addition, infant formula producers must accomplish guidelines aimed to decrease the risks of products contamination with foodborne pathogens. Controlling the primary populations of E. cowanii during the PIF production process and preventing post processing contamination by using suitable microbiological guidelines, is accessible. Sanitary practices for the preparation of infant formula in both the home and hospitals should be carefully controlled.

Conclusion
Powdered infant milk formula containing members of the Enterobacteriaceae might impose additional risk of infection to the neonates and especially to the low birth weight premature babies. There is very little information about virulence factors and pathogenicity of E. cowanii in human, so complementary studies are necessary to clarify the possible role of E. cowanii as a food contaminant, in common NICU infections and high risk groups including persons with underlying disease and immunocompromised individuals.